1. Field of the Invention
The invention relates generally to the field of cellular biology. More particularly it concerns the use of a laser scanning cytometer with a digital camera to capture an image of cells in a sample for diagnosis of cell characteristics. More specifically, it relates to diagnostics regarding cancerous cells.
2. Description of Related Art
Pre-malignant and malignant cellular transformation is associated with changes in cell morphology and in DNA content. For early cell transformations in the absence of apparent aberrant cell morphology (a negative cytology), changes in DNA content, detected by ploidy analysis, are critical in establishing the diagnosis. DNA ploidy analysis is performed using a Laser Scanning Cytometer (LSC(trademark)). The LSC is laser based technology that combines immunofluorescence, cell morphology and DNA ploidy analysis to enhance the sensitivity and specificity for detecting malignant cells. This method is capable of not only measuring DNA ploidy but identifying individual cell types based on their immunophenotype. The immunophenotype of epithelial cells from urine is determined by staining the cell cytoplasm for the presence of cytokeratin. By combining the immunophenotype with the DNA ploidy pattern, the pathologist can now identify epithelial cells that are aneuploid, a profile consistent with specific cancer cells.
Previous methods using laser scanning cytology, such as described in U.S. Pat. No. 5,793,969, U.S. Pat. No. 5,885,840, U.S. Pat. No. 5,427,910 and Kawamura et al. (2000) are directed to LSC methods in which the images are visualized by a monitor display or by means of a CCD camera, and stored in a computer disk file. This arrangement of equipment is most useful if a cytopathologist is on-site and furthermore present during analysis to visualize the image on the monitor and make the diagnosis at that point in time. The image itself is additionally not transferable to others related to its diagnosis. Thus, a method to remove this constraint for analysis and moreover provide a tangible means to retain the data is lacking in the art.
The present invention described herein overcomes significant limitations in the art of laser scanning cytology methods. The inventors describe herein methods to record data from a biological sample containing a cell by using a digital camera. In specific embodiments, the capture of the image in a digital form facilitates examination of the data in a format conducive to a more thorough analysis and diagnosis. The inventors envision that the LSC methods described herein permit a more efficient and convenient analysis of immunophenotype, DNA ploidy and cell morphology in pursuit of diagnosis of cell characteristics, such as those related to cancer. In further specific embodiments, the analysis of the cell characteristics from a urine sample using the LSC methods of the present invention provide diagnosis for the presence of transitional cell carcinoma.
In an embodiment of the present invention there is a method for recording cell sample data comprising providing a sample containing a cell; subjecting said sample to a laser-based interrogation; and capturing a digital image of one or more cell characteristics. In a specific embodiment, the digital image is directly captured. In another specific embodiment, the method further comprises analyzing one or more cell characteristics. In an additional specific embodiment, the method further comprises analyzing one or more cell characteristics by fluorescent in situ hybridization. In a further embodiment, the one or more cell characteristics are selected from the group consisting of immunophenotype, DNA ploidy, cell morphology, nuclear fragments and subcellular fragments. In a specific embodiment, one or more cell characteristics comprises immunophenotype, DNA ploidy, cell morphology, nuclear fragments and subcellular fragments. In an additional embodiment, the cell is selected from the group consisting of an animal cell, a plant cell and a prokaryotic cell. In another specific embodiment, the animal cell is selected from the group consisting of a cancer cell, an epithelial cell, a bone marrow cell, a red blood cell, a white blood cell, a muscle cell, a bone cell, a connective tissue cell, a nerve cell and a brain cell. In another embodiment, the cell is a cancer cell. In a further specific embodiment, the method further comprises the step of preparing a cell sample. In a specific embodiment, the preparation step comprises processing of a sample, wherein said sample processing is selected from the group consisting of concentration, dilution, microscope analysis preparation, applying a stain or marker agent, or a combination thereof. In an additional specific embodiment, the sample is selected from the group consisting of urine, blood, sputum, feces, saliva, marrow, touch preps of surgical specimens, fine needle aspirates and cellular body fluid.
In an additional embodiment of the present invention the method further comprises capturing a video image, a color image or a black and white image of said cell sample. In another specific embodiment, the marker agent is selected from the group consisting of cytokeratin, propidium iodide, CD19, CD34, CD3, annexin V, and a combination thereof. In another specific embodiment the DNA ploidy of said cell is aneuploid. In another specific embodiment, one or more cell characteristics are analyzed from the same said digital image. In a further specific embodiment the immunophenotype is determined by applying a marker agent. In another specific embodiment, the marker agent is selected from the group consisting of cytokeratin, propidium iodide, CD19, CD34, CD3, annexin V, and a combination thereof. In an additional specific embodiment, the immunophenotype is determined by applying multiple marker agents.
In another object of the present invention there is a method of diagnosing cancer comprising providing a sample containing a cell; subjecting said sample to a laser-based interrogation; capturing a digital image of one or more cell characteristics; and identifying aneuploidy based on said one or more cell characteristics.
In an additional object of the present invention there is a laser-based cell analyzer comprising a laser; a first camera port; a second camera port; and a third camera port, wherein said cell analyzer facilitates determination of cell type. In a specific embodiment, the cell analyzer further comprises a digital camera attached to one camera port. In another specific embodiment, the cell analyzer further comprises a color camera attached to one camera port. In an additional embodiment, the cell analyzer further comprises a black and white camera attached to one camera port.
In another object of the present invention there is a laser-based cell analyzer system comprising a black and white camera attached to a first camera port; a color camera attached to a second camera port; and a digital camera attached to a third camera port. In a specific embodiment, the cell analyzer system further comprises a computer. In an additional embodiment, the cell analyzer system further comprises a printer.
In an additional object of the present invention there is a method for creating a cell data record comprising subjecting a cell to laser interrogation; and recording in a tangible medium one or more cell characteristics in a digital format.